Tracing Breast Cancer

Angiogenesis-promoting Proteins May Signal Risk of Disease

The kidneys squeeze out more than a liter of urine each day. Over the past 13 years, Marsha Moses and her colleagues have been panning the amber liquid for proteins and other particles that might indicate the presence of cancer and other disease. In 1998 they pulled out a set of angiogenesis-promoting proteins, the matrix metalloproteinases (MMPs), from the urine of people with a variety of cancers. A few years later, they set themselves an even more ambitious challenge: could they tell, just from urine, who might develop a particular type of cancer in the first place? The results are finally in and it appears that urine-just a few drops-can reveal which women may be at risk for breast cancer.

"We really wanted to push the envelope and think about risk assessment," said Moses, HMS professor of surgery at Children's Hospital Boston. Working with Susan Pories and colleagues, Moses analyzed urine from women harboring two kinds of breast lesions, each associated with a higher risk for cancer. Moses and her colleagues found that the women's urine exhibited much higher levels of MMP-9 and a related enzyme, ADAM-12, than did that of controls.

The study, which appears in the May 22 Cancer Epidemiology, Biomarkers and Prevention, and other findings coming out of the Moses lab are helping to put a radically new spin on the time-honored practice of collecting and analyzing urine. Borrowing methods from the burgeoning field of proteomics, such as mass spectrometry, researchers have been scouring human urine for scraps of protein and other disease-related molecules. RNA and DNA have already been found.

Practitioners of the new field of urine proteomics have been talking about using urine dipsticks to do the work of more invasive or expensive techniques-phlebotomists' needles and PET and MRI scanners-to diagnose and monitor the progression of cancer and other diseases. The findings by Moses, Pories, Roopali Roy, David Zurakowski (an assistant professor of orthopedic surgery at Children's), and colleagues suggest urine might have even greater power-the ability to spot disease before it actually happens.

Warning Signs

In retrospect, the MMPs appear well suited to the task. To take root, cancer cells must carve through the surrounding extracellular matrix, and they do so by secreting MMPs. As a tumor grows in size, it must release even more MMP-and, indeed, Moses and her colleagues have found that levels of excreted MMP and ADAM-12 can be used to measure cancer progression.

Finally, tumors inhabit different extracellular environments and may call upon a very specific set of MMPs. Roy, HMS instructor in surgery at Children's, Moses, and colleagues recently analyzed the urine of patients with bladder and prostate cancer and found that they could distinguish the two cancers based on their constellation of MMPs.

"There appears to be a tumor-specific fingerprint," said Moses, who, with Roy and colleagues, will soon be publishing the findings. What this means is that doctors might someday be able to tell from urine where in the body a tumor is located by looking at the MMP fingerprint.

Yet until recently, many researchers dismissed the MMPs for a simple reason: they were thought to be too massive to pass through the kidneys.

Indeed, when Moses and her colleagues set out, the idea of looking for urinary MMPs seemed quixotic. Having worked extensively with the enzymes in the early 1990s, however, she thought it was at least worth a try. Using simple gel electrophoresis, she and colleagues scoured samples from over 140 subjects with cancers of all sorts. They found the bulky molecules. "They were there, big as day," said Moses.

Using tandem mass spectrometry, they went on to identify even more cancer biomarkers, which they then validated against their growing database of thousands of urine samples. (Moses and colleagues have created the largest institutional review board-approved human urine bank in the country.) One of these, a metalloproteinase called ADAM-12, caught their attention. "This one just stood out so strongly in our breast cancer patients," she said. In 2006, Pories, an HMS assistant professor of surgery, Moses, and colleagues began analyzing the urine of 68 women with breast cancer risk-marker lesions-44 with atypical hyperplasia and 24 with lobular carcinoma in situ-and 80 healthy controls. Over 75 percent of lesion-carrying women exhibited high levels of ADAM-12, compared with four percent of controls. MMP-9 was also much more prevalent in the affected population.

Moses, Pories, and colleagues found the enzyme's ability to identify at-risk women was even greater when combined with a commonly used clinical tool, called the Gail score, which evaluates aspects of a woman's history such as age, alcohol, and cigarette use, and family history of breast cancer.

Gentler Tests

Children's Hospital has granted a license to develop the MMP, ADAM-12, and other urinary biomarkers to a company, Predictive Biosciences, cofounded by Moses and Bruce Zetter, the Charles Nowiszewski professor of cancer biology in the Department of Surgery at Children's. The company is currently using the technology to develop urine tests that would detect the recurrence of bladder and other cancers. Currently, physicians must resort to invasive procedures to monitor patients after treatment. "A noninvasive approach would be a godsend for these patients," said Moses. It could also allow doctors to see how their patients are doing soon after treatment. Or, if a woman's urine revealed that she was at risk for breast cancer, she might opt for more frequent mammograms. She might quit smoking, drinking alcohol, taking exogenous estrogens and might make positive lifestyle changes such as eating less fat and getting more exercise.

That might seem a tall order for a liquid that, medically, has been seen as a poor cousin to tissue and blood-able to diagnose pregnancy, diabetes, and kidney disorders but not far-flung cancers. But some believe a new breed of urine tests could play a critical role in the future of personalized medicine.

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